Live Imaging of Phagosome Migration in HIV-1-Infected Human Macrophages

Place an imaging dish containing human monocyte-derived macrophages expressing Fc receptors onto the confocal microscope stage for live imaging.

These macrophages are pre-infected with an HIV-1 strain expressing green fluorescent protein or GFP.

Identify and focus on a single GFP-positive macrophage to ensure clear visualization.

Remove the dish and add IgG-opsonized sheep red blood cells or SRBCs to the macrophages.

Briefly centrifuge the dish to facilitate interaction between Fc receptors on the macrophages and IgG on the SRBCs, enabling internalization and phagosome formation.

Return the dish to the microscope and refocus.

Set the Z-stack imaging parameters and acquire both fluorescent and bright field images of the cell.

Use the fluorescent channel to identify GFP-positive HIV-infected cells, and the bright field channel to track the movement of internalized, non-fluorescent phagosomes.

This method enables real-time visualization of phagosome migration in HIV-infected macrophages.

Place the imaging dish on the microscope stage. Adjust the focus and the location to find only one whole HIV-1-infected macrophage in the field.

Use appropriate excitation and emission settings based on the used imaging system and probe. Include a bright field channel. Optimize its appearance by adjusting the exposure time.

Next, remove the imaging dish and add one milliliter of sheep red blood cell, or SRBC suspension, at seven times tenth to the sixth SRBCs per milliliter, to the dish. Centrifuge the dish at 500 times g for two minutes at room temperature to synchronize phagocytosis, then record the time at the end of the centrifugation, and return the dish to the stage. Optimize the focus and set the top and bottom of the stack with a step distance of 0.3 micrometers.

Start acquisition of the Z stacks of GFP and BF images throughout the thickness of the cell, and save the time-lapse video in the native file format of the imaging system.